Complete deficiency of the enzyme hypoxanthine phosphoribosyltransferase (HPRT) in humans causes Lesch-Nyhan syndrome, an X-linked neurological disorder characterized by mental retardation, choreoathetosis, self- mutilative behavior and hyperuricemia. Partial deficiency causes a severe form of gouty arthritis and approximately 20% of these patients have mental retardation and spasticity. HPRT is considered a "housekeeping" gene as it is constitutively expressed at low levels in all tissues. However, it is expressed at five- to seven-fold higher levels in parts of the central nervous system. To understand the role of cis-acting sequences in the tissue-differential expression of the human HPRT gene, we have functionally analyzed 1600 bp of the 5' flanking region in cultured cells and in transgenic mice. We have identified a 182 bp negative regulatory element (hHPRT-NE) upstream of the core promoter which represses expression in cultured fibroblasts. Transgenic mice studies have shown that the hHPRT-NE is one of at least two elements that direct transgene expression in the forebrain of the mouse. We have found that the core promoter of 97 bp is apparently not sufficient for constitutive expression in transgenic mice. We have identified trans-acting factors interacting with hHPRT-NE, both ubiquitous and brain-specific. This application proposes to identify trans-acting factors from tissues such as brain, liver and kidney, that interact with the cis-acting regulatory sequences of the human HPRT gene and are important for maintaining tissue-differential expression of the gene. The interaction of these trans-acting factors with these sequences will be characterized at the nucleotide level. cDNA clones expressing these factors will be isolated and used for their expression in bacterial cells and in mammalian cells, to allow authentication and the generation of antibodies, and for functional analysis, respectively. The tissue- distribution of the trans-acting factors will be studied during development in relation to that of HPRT gene expression. These studies will support and confirm our ongoing studies on functional delineation of cis-acting sequences important for tissue-differential expression of the HPRT gene. They will also define factors important for "housekeeping" and tissue-differential gene expression. HPRT deficiency is an excellent candidate for gene therapy. The information provided by this study will, by defining the sequences and factors required for appropriate patterns of expression of the HPRT gene, prove very valuable in designing vectors for such therapy.